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Genetic toxicity in vitro

Description of key information

In vitro, the genetic toxicity of isopentyl p-methoxycinnamate was investigated in a series of 4 Ames tests in the S. typhimurium tester strains TA98, 100, 1535, 1537, and 1538 in concentrations ranging from 3 to 150 µL/plate or 5,000 to 25,312.5 µg/plate. In addition an in vitro mutagenicity test was performed in mammalian cells (CHO cells) and was considered the key sudy for mutagenicity. Additionally, isopentyl p-methoxycinnamate was tested in a chromosomal aberration test in human peripheral blood lymphocytes, both in the presence and absence of metabolic activation by rat S9 mix.

Genetic toxicity in vivo

Description of key information

As a consequence of the positive result in one of the Ames tests, the genetic toxicity of isopentyl p-methoxycinnamate was further studied in the in vivo micronucleus test in mice due the requirement under REACH Annex VIII.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-02-11 to 1986-03-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The study was conducted to a method comparable to OECD guideline no. 474. However, it is not stated anywhere in the study report that the study was conducted in accordance with any guideline. The study was conducted in accordance with the principles of GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 1
Principles of method if other than guideline:
The micronucleus test is a mammalian in vivo test which detects damage to the chromosomes and the mitotic apparatus induced by chemicals.
The test involves the microscopic examination of cytological preparations of polychromatic erythrocytes obtained from the bone marrow of treated and control animals. An increased frequency of micronucleated polychromatic erythrocytes among treated animals compared to control animal values is taken as an indication of treatment-induced genetic damage.
Any toxic effects of the test substance in the immature nucleated cells may lead to a reduction of cell division and cell death. This creates a void in the marrow canal. The empty space is then filled with peripheral blood (Von Ledebur and Schmid, 1973). If the ratio of polychromatic to normochromatic erythrocytes is determined and found to be significantly different from the control value, this is taken as indication of cytotoxicity.
In the present study, the compound was tested in the micronucleus test using both male and female NMRI mice. Three dosages and with the highest dose three times of preparation were investigated to obtain an overview on possible genetic effects.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Adult male and female NMRI mice, 25-30 g in weight and 6-10 weeks old were obtained from SAVO-IVANOVAS GmbH (Kisslegg, Germany).
The animals were acclimatised for at least 5 days before use in the test. They were examined for signs of disease. Animals suspected of being diseased were culled from the study and replaced by others. The animals were separated according to sex, marked for dentification, and allocated by randomization to cages and groups.
The animals were group housed in groups of up to five animals per cage in MAKROLON cages of size II with ALTROMIN wood shaving. The air-conditionned surrounding had a temperature of 22±2°C and a relative humidity of ca. 55%.
Artificial light was provided in a 12/12 h light/dark cycle. The animals were given standard laboratory diet (ALTROMIN 1324, Altromin, Lage, Germany) and tap-water ad libitum. The cages and beddings were changed with clean ones twice a week.
Route of administration:
intraperitoneal
Vehicle:
The compound was dissolved in oilve oil. The solution was freshly prepared just prior to use.
Details on exposure:
Three groups, each comprising 5 males and 5 females, were treated with a low, intermediate or a non-lethal, highly toxic dose of the test compound (5 animals per group and sex and term)
Duration of treatment / exposure:
A dose volume of 10 mL/kg was administred intraperitoneally by injection.
Frequency of treatment:
The animals were dosed once (at 0 h).
Post exposure period:
Sampling was performed at 24 h for the low, intermediate, and high dose. 2 additional groups were treated with the high dose and preparations were made at 48 and 72 h.
Remarks:
Doses / Concentrations:
3000
Basis:
other: mg/kg body weight
Remarks:
Doses / Concentrations:
1500
Basis:
other: mg/kg body weight
Remarks:
Doses / Concentrations:
750
Basis:
other: mg/kg body weight
No. of animals per sex per dose:
Each group (per dose) comprised 5 males and 5 females.
Control animals:
yes, concurrent vehicle
Positive control(s):
A positive control group, also consisting of 5 males and 5 females, was treated with cyclophosphamide at a dose level of 50 mg/kg. The animals of both control groups were killed at 24 h after treatment. Cyclophosphamide (batch 3006, Asta-Werke, Bielefeld, Germany), was dissolved in phosphate buffered saline.
Tissues and cell types examined:
The animals were killed by cervical dislocation. Bone marrow was removed from both femora by rinsing with foetal calf serum. Bone marrow cells were centrifuged at 150 g for 10 min. and the supernatant was discarded. From the pellet, smears were made on slides and air-dried, according to haematological routine. Two slides were made per animal.
Details of tissue and slide preparation:
The preparations were stained by the May-Gruenwald-Giemsa method according to Schmid (1973):
- Stained for 3 min in undiluted May-Gruenwald solution
- Stained for 2 min in May-Gruenwald, diluted with distilled water 1:1
- Rinsed briefly in distilled water
- Stained for 10 min in Giemsa, diluted with distilled water 1:6
- Rinsed thoroughly in distilled water
- Air-dried, the back side of the slides were cleaned with methanol
- Cleared with xylene for 5 min, and mounted in Eukitt
Evaluation criteria:
If the ratio of polychromatic to normochromatic erythrocytes is determined and found to be significantly different from the control value, this is taken as indication of cytotoxicity.
Statistics:
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970)
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
no additional infomation available

After dosing, the animals of all groups treated with the test substance showed a reduced motility and piloerection. The animals of the highest dosage showed the most pronounced effect. In the animals of all groups, the ratio of polychromatic to normochromatic erythrocytes was normal.

Conclusions:
The results indicate that isopentyl p-methoxycinnamate does not induce chromosomal damage to the mitotic apparatus in bone marrow cells of mice.
Executive summary:

The mutagenic effect of the compound isopentyl p-methoxycinnamate was studied by means of the micronucleus test in bone marrow cells of NMRI mice.

Male and female mice were treated with one intraperitoneal injection of the test substance isopentyl p-methoxycinnamate in olive oil at dose levels of 750, 1500, and 3000 mg/kg and a standard volume of 10 ml/kg. Bone marrow smears were prepared at 24 h after treatment. With the highest dosage, additional preparations at 48 and 72 h were made. Each group comprised 5 males and 5 females.

A vehicle control group, consisting of 5 males and 5 females, was dosed with olive oil. A positive control group, also consisting of 5 males and 5 females, was treated with cyclophosphamide at a dose level of 50 mg/kg. The animals of both control groups were killed 24 h after treatment.

At least 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes was also recorded. The ration of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Under the test conditions described, the test compound isopentyl p-methoxycinnamate failed to induce a significant increase in micronucleated polychromatic erythrocytes compared with the negative controls at any dose tested. The positive control substance, cyclophosphamide, however, produced a huge increase in the frequency of micronucleated polychromatic erythrocytes.

The results indicate that isopentyl p-methoxycinnamate does not induce chromosomal damage to the mitotic apparatus in bone marrow cells of mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro Mutagenicity tests:

The mutagenicity of isopentyl p-methoxycinnamate was investigated in a series of 4 Ames tests in the S. typhimurium tester strains TA98, 100, 1535, 1537, and 1538 in concentrations ranging from 3 to 150 µL/plate or 5,000 to 25,312.5 µg/plate. All tests were negative with the exception of an isolated positive finding obtained in one Ames test in tester strain TA100 in the absence of metabolic activation (study no. 1987025). It is considered that this particular test is irrelevant, as all other Ames tests, including a re-test in TA100 in the absence of metabolic activation conducted in the same testing laboratory (study no. 1989139), were clearly negative. Precipitating concentrations were reached in each case and all tests were carried out both in the presence and absence of metabolic activation by rat S9 mix.

In addition an in vitro mutagenicity test was performed in mammalian cells (CHO cells) and was considered the key study for mutagenicity as this study confirmed the negative status for a mutagenic response. Chromosome aberration test: Isopentyl p-methoxycinnamate was tested in a chromosomal aberration test in human peripheral blood lymphocytes, both in the presence and absence of metabolic activation by rat S9 mix. The test concentrations were 20, 100, and 300 µg/mL in the test with metabolic activation, and 10, 30, and 100 µg/mL in the test without metabolic activation. Isopentyl p-methoxycinnamate was negative in this test. In vivo genetic toxicity: The genetic toxicity of isopentyl p-methoxycinnamate was studied in the in vivo micronucleus test in mice. Male and female NMRI mice (5 males and 5 females per group) received an intraperitoneal injection of isopentyl p-methoxycinnamate in olive oil at dose levels of 750, 1,500, and 3,000 mg/kg, or the vehicle only. Cyclophosphamide (50 mg/kg) was used as positive control. Bone marrow smears were prepared at 24 h after treatment. With the highest dosage, additional preparations at 48 and 72 h were made. Also in this test, isopentyl p-methoxycinnamate was clearly negative.

Negative results were obtained from the in vitro chromosomal aberration test (King 1991), the in vitro mammalian cell gene mutation assay (Hall 2010), and from all except one of the four Ames tests (Herbold 1983; Insley 1980; King 1987, 1989). Furthermore, isopentyl p-methoxycinnamate was negative in the in vivo micronucleus test in mice. Accordingly, the results of the genetic toxicity studies indicate that isopentyl p-methoxycinnamate is devoid of any genotoxic activity.

Justification for classification or non-classification

Based on the results of the in vitro and in vivo genetic toxicity assays outlined above, it can be concluded that isopentyl p-methoxycinnamate is non-genotoxic. Accordingly, since the absence of any genotoxic effects has been shown in bacteria and in somatic cells in vitro as well as in mice in vivo, isopentyl p-methoxycinnamate is not classified as a germ cell mutagen as specified in the current EU-CLP Regulation. However, results from specific mutagenicity studies in germ cells are lacking.